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I'm interested if there is a way to degrade the plasmid DNA inside an E.Coli cell specifically so that the method does no harm to the chromosomal DNA. First I was thinking about restriction endonucleases but I'm afraid that;

  • it isn't specific enough and kills the cell
  • or it does nothing, because the E.Coli methylates the plasmid DNA too

My second thought was the CRISPR/Cas9 system but it sounds like an overcomplication of the problem. Any simpler ideas? (For eg. specific enzymes)

EDIT: the main idea is to create more competent E.Coli cells with directed evolution. Currently this is just a thought experiment, so there is no specific plasmid sequence. In details; I would transform the plasmid into the cells, then do the selection part with an antibiotic and let them grow, then transfer the cells into a medium where there is no antibiotic and add IPTG to induce the plasmid-DNA-hydrolase enzyme, then maybe an other selection part could be performed to remove the cells which still contain the plasmid (for example with FACS if there is a GFP gene on the plasmid), then make the cells competent again with CaCl2 and do this whole process over and over again.

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  • $\begingroup$ There are a lot of (simple) solutions, but I'm not really sure what you want to achieve. Can you clarify? $\endgroup$ – VonBeche Oct 27 '16 at 8:24
  • $\begingroup$ Why do you want to do this? To get rid of the plasmid and re-use the cells for something else? $\endgroup$ – Chris Oct 27 '16 at 8:30
  • $\begingroup$ @Chris Exactly, the core idea was that if such an enzyme exists then I could elevate the competitive ability of the cells by directed evolution. $\endgroup$ – fazekaszs Oct 27 '16 at 10:11
  • $\begingroup$ Do you know the sequence of the plasmid? It would not be straightforward to get rid of the plasmid especially if it is high-copy number. $\endgroup$ – WYSIWYG Oct 27 '16 at 17:12
  • $\begingroup$ @VonBeche I tried to clarify it in the question details. What are those simple solutions? $\endgroup$ – fazekaszs Oct 28 '16 at 7:51
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traditional method to remove a plasmid from E. Coli is to grow the cells with Etidium bromide.

http://vlab.amrita.edu/?sub=3&brch=186&sim=1097&cnt=1

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  • $\begingroup$ Done. But really... this is like needing to reference plasmid extraction by alkaline lysis or pouring an agarose gel. $\endgroup$ – JayCkat Dec 19 '16 at 5:29

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